Transcriptional regulation of anthocyanin
biosynthesis in a high-anthocyanin
, Ujjal Kumar Nath1,2†
, Jong‑In Park1*
, Mohammad Rashed Hossain1,2
Manosh Kumar Biswas1
, Hoy‑Taek Kim1,3
, Hye Ran Kim4
and Ill‑Sup Nou1*
Background: Anthocyanins are plant secondary metabolites with key roles in attracting insect pollinators and protecting against biotic and abiotic stresses. They have potential health‑promoting effects as part of the human diet. Anthocyanin biosynthesis has been elucidated in many species, enabling the development of anthocyanin‑enriched fruits, vegetables, and grains; however, few studies have investigated Brassica napus anthocyanin biosynthesis.
Results: We developed a high‑anthocyanin resynthesized B. napus line, Rs035, by crossing anthocyanin‑rich B. rapa
(A genome) and B. oleracea (C genome) lines, followed by chromosome doubling. We identified and characterized 73 and 58 anthocyanin biosynthesis genes in silico in the A and C genomes, respectively; these genes showed syntenic relationships with 41 genes in Arabidopsis thaliana and B. napus. Among the syntenic genes, twelve biosynthetic and six regulatory genes showed transgressively higher expression in Rs035, and eight structural genes and one regula‑ tory gene showed additive expression. We identified three early‑, four late‑biosynthesis pathways, three transcrip‑ tional regulator genes, and one transporter as putative candidates enhancing anthocyanin accumulation in Rs035. Principal component analysis and Pearson’s correlation coefficients corroborated the contribution of these genes to anthocyanin accumulation.
Conclusions: Our study lays the foundation for producing high‑anthocyanin B. napus cultivars. The resynthesized lines and the differentially expressed genes we have identified could be used to transfer the anthocyanin traits to other commercial rapeseed lines using molecular and conventional breeding.
Keywords: Brassica napus, Resynthesized, Microsynteny, Transgressive expression, Additive expression, Anthocyanins
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Anthocyanins belong to the flavonoid class of second-ary metabolites. These water-soluble pigments are widely distributed in plants, accumulating in the leaves, petals, sepals, and fruits to yield purple, red, and blue coloration [1, 2]. Anthocyanins attract insects for pollination and protect plants against biotic and abiotic stresses [3–7]. Eating foods enriched with anthocyanins might reduce inflammation and protect against certain types of cancer,
cardiovascular, neurodegenerative, and various age-related diseases [8–10]. The health benefits of anthocya-nins are believed to be closely linked to their antioxidant activities, through which they reduce the abundance of reactive oxygen species . Anthocyanins also increase blood serum antioxidant levels and protect red blood cells from oxidative damage [8, 12].
Brassica napus is a natural allopolyploid with the genomic composition 2n = 4x = 38 (AACC). In addition to its international use as an oilseed and fodder crop, it is becoming increasingly popular as a vegetable, and a number of B. napus vegetable varieties have been devel-oped . For example, the leaves and edible flower buds and young stems of B. napus are consumed as vegetables
*Correspondence: email@example.com; firstname.lastname@example.org
†Gayatri Goswami and Ujjal Kumar Nath contributed equally to this work 1 Department of Horticulture, Sunchon National University, 255
in China and other Asian countries, and one subspecies is also commonly used as a root vegetable in Europe, the U.S., Canada, and Australia (rutabaga, B. napus subsp. rapifera). Considering the diverse health-promoting benefits of anthocyanins, B. napus cultivars with high anthocyanin contents may be popular with consum-ers; however, only one study has documented B. napus mutants with red or purple leaves . In contrast, sev-eral high-anthocyanin varieties are available for two of the most popular leafy Brassicaceae vegetables, Chinese cabbage (B. rapa ssp. pekinensis) and cabbage (B. olera-cea var. capitata).
Understanding the regulation of anthocyanin biosyn-thesis is vital for enabling us to develop anthocyanin-enriched fruits and vegetables. Anthocyanin biosynthesis begins in the cytoplasm under the coordinated regu-lation of the structural genes, which encode several enzymes and regulatory transcription factors . Glu-tathione S-transferases are involved in the export of the anthocyanins from the cytoplasm to the vacuole, where they are permanently stored [16, 17]. The anthocyanin
biosynthesis pathway can be divided into three major consecutive phases (Fig. 1). In the first phase, phenyla-lanine is converted into trans-cinnamic acid and 4-cou-maroyl-CoA by phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), respectively [18–20]. Next, a series of enzy-matic reactions catalyzed by chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) lead to the formation of dihydroflavonol from one molecule of coumarate-CoA and three molecules of malonyl-CoA. In the third stage, many types of antho-cyanidins are formed from the dihydroflavonols, in reac-tions catalyzed by dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). Anthocyanidins are finally modified by a series of glycosylation and meth-ylation steps to form anthocyanins, catalyzed by UDP-glucose:flavonoid glucosyltransferase (UFGT) and methyl transferase (MT) [21, 22].
Both structural and regulatory genes are involved in anthocyanin accumulation in Arabidopsis thaliana [19, 23, 24]. Primarily, anthocyanin biosynthesis is regulated
FLS UFGT Flavonol glycosides ( no color or pale
FNS UFGT Flavone glycosides
( no color) Phenylalanine
4-coumaroyl CoA 4-coumaric acid
(pink or red)
3x malonyl CoA (CHS)
Vacuolar transport (AATs) Early biosynthetic
Cyanidin 3-diglucoside-5-glucoside, cyanidin 3- (sinapoyl)-diglucoside-5-glucoside, cyanidin 3-sophoroside-5-malonlyglucoside
Malvidin-3-(p-coumaroyl)glucoside pyruvic derivative
cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside
Fig. 1 Schematic and simplified diagram of the anthocyanin biosynthesis pathway leading to the accumulation of anthocyanin in Brassica. The pathway is divided into three major parts; the upstream phenylpropanoid pathway, the early biosynthetic pathway, and the late biosynthetic pathway, based on the types of genes involved in each stage. The possible activation sites of the regulatory MBW complexes are indicated. Details of the genes involved in each stage are provided in Table 2. PAL phenylalanine ammonia lyase, C4H cinnamate‑4‑hydroxylase, 4CL 4‑coumaratel‑CoA ligase, CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone‑3‑hydroxylase, FLS flavonol synthase, DFR dihydroflavanol reductase, ANS
at the transcriptional level; previous studies demon-strated that the expression patterns of the anthocyanin structural genes are mainly determined by the R2R3-MYB (myeloblastosis family of transcription factors), basic helix-loop-helix (bHLH), and WD40-repeat tran-scription factors, and by their coordinated interactions . Lang et al.  reported that R2R3-MYB and bHLH transcription factors can bind to specific cis-acting ele-ments in promoter regions of the structural genes and regulate their expression. The MBW (R2R3-MYB, bHLH, and WD40) complex is unique to plants, but this tran-scriptional regulatory system may vary between monocot and dicot plants; all three members of the MBW complex are not required for anthocyanin biosynthesis in every species [21, 22].
Recent studies have revealed that, in addition to the regulatory genes, the structural genes could also play a vital role in the accumulation of high levels of antho-cyanin in several plant species [18, 27]. Zhang et al.  reported that most of the structural genes, including PAL,
CHS, F3H, F3′H, DFR, ANS, and anthocyanin
5-0-gluco-syltransferase (5GT) were significantly upregulated in purple kohlrabi (Brassica oleracea var. gongylodes), while only two regulatory genes, transparent testa8 (TT8)
and production of anthocyanin pigment2 (PAP2) were
upregulated. Previous reports showed that most of the anthocyanin pathway structural genes are upregulated in B. rapa varieties with high anthocyanin levels [28, 29]. The increased expression of structural genes in red cab-bage was found to be coordinated by a bHLH, TT8, and a MYB transcription factor gene, MYB2 .
Allopolyploidization, the merging of two or more divergent genomes into a common background, enhances the genomic diversity of the resulting organ-ism [30–32]. Allopolyploids arise from interspecific or intergenic hybridization followed by chromosome dou-bling, and can act as a bridge species for gene introgres-sion into important crop species. The genus Brassica contains a number of diploid and allopolyploid species. Among them, six agriculturally important species can be categorized into three basic diploid genomes (A, B, and C; n = 10, 8, and 9, respectively) and their allopoly-ploid hybrids (AB, AC, and BC), as confirmed using cytogenetics . Brassica napus (AACC; 2n = 38) is an allopolyploid that evolved following the hybridization of its ancestors B. rapa (AA; 2n = 20) and B. oleracea (CC, 2n = 18) . Sequence variations within chromosome segments showed that some of the A- and C-genome components of B. napus have undergone genetic changes since this hybridization event [34–36]. In contrast, resyn-thesized B. napus (produced by crossing B. rapa and B. oleracea followed by embryo rescue and chromosome doubling) was shown to undergo extensive chromosomal
rearrangements immediately after allopolyploidization [37–39]. In the present study, we therefore found a wide variation in anthocyanin contents in resynthesized B. napus lines rather than either of the parents or interme-diate of the parental lines.
Here, we characterized the structural and regulatory genes of anthocyanin biosynthesis in a purple-colored resynthesized B. napus line. We developed an antho-cyanin-enriched allopolyploid B. napus by crossing a red-green Chinese cabbage (B. rapa ssp. pekinensis, AA genome) and a red cabbage (B. oleracea var. capitata, DD genome), followed by embryo rescue and chromosome doubling using colchicine. The aims of this study were to (1) select a stable anthocyanin-enriched resynthesized B. napus line developed by interspecies hybridization, (2) investigate the impact of the total anthocyanin con-tent on the purple pigmentation of the leaves, (3) predict the candidate genes responsible for the purple leaf trait in B. napus using expression profiling and determine the expression patterns of those genes, and (4) determine the relationship between the different gene expression pro-files and total and constituent anthocyanin contents in B. napus.
Selection of an anthocyanin‑enriched resynthesized B. napus line
The anthocyanin-enriched B. napus line was selected based on visual observations and spectrophotometric of the 39 resynthesized (Rs) lines developed by cross-ing a red-green Chinese cabbage inbred line (Asia) and a red cabbage inbred line (Rubea) followed by embryo rescue and chromosome doubling. Allopolyploids result-ing from the interspecies cross were confirmed usresult-ing the COS1078 marker and by checking their DNA contents using flow cytometry (data not shown). The Rs lines had a wide variety of total anthocyanin contents (< 0.5 to > 4.5 mg g−1 FW) (Fig. 2). Five Rs lines with total
antho-cyanin contents > 4.0 mg g−1 FW were selected and
eval-uated in a five-replicate trial during the next season. Of these, Rs035 had the highest total anthocyanin content (5.13 ± 0.17 mg g−1 FW) and was selected for further
expression profiling using qRT-PCR.
Evaluation of the anthocyanin profiles in Brassica lines
oleracea parent (cv. Rubea) (Fig. 2). No anthocyanin was detected in the resynthesized green B. napus line Rs306.
We carried out HPLC to investigate the anthocyanin composition of our contrasting Brassica plants. A total of six anthocyanin components were detected in the four lines (Table 1), of which cyanidin-3-diglucoside-5-glucoside, cya-nidin-3-(sinapoyl)-diglucoside-5-glucoside, cyanidin-3-so-phoroside-5-malonylglucoside, malvidin-3-(p-coumaroyl) glucoside pyruvic derivative, and pelargonidin-3-(6-acetyl)-glucoside were the major anthocyanin components in Rs035. Four anthocyanin components were detected in the Chi-nese cabbage line Asia: cyanidin-3-diglucoside-5-glucoside, 3-(sinapoyl)-diglucoside-5-glucoside,
cyanidin-3-sophoroside-5-malonylglucoside, and pelargonidin-3-(6-acetyl)-glucoside. Among the six anthocyanin components, only three components, cyanidin-3-diglucoside-5-glucoside, cyanidin-3-(sinapoyl)-diglucoside-5-glucoside, and cyani-din-3-(p-coumaroyl)-diglucoside-5-glucoside, were found in the red cabbage line Rubea. None of the anthocyanin com-ponents were detected in the green Rs306 line (Table 1).
Selection and in silico characterization of anthocyanin biosynthesis pathway genes
To predict and characterize the putative anthocyanin biosynthesis candidate genes in resynthesized B. napus, the anthocyanin pathway genes in A. thaliana were used 0
1 2 3 4 5 6
a Rs 00
Total anthocyanin conten
B. rapa parent, Asia
B. oleracea parent, Rubea
B. napus, resynthesised lines
Fig. 2 Total anthocyanin contents in 39 resynthesized B. napus lines along with their parental lines
Table 1 Anthocyanin composition in leaves of B. rapa reddish line Asia (A-genome), resynthesis green B. napus line Rs306, resynthesis red B. napus line Rs035 and B. oleracea red cabbage line Rubea (C-genome) as detected by HPLC
ND not detected
Anthocyanin component Anthocyanin levels (mg g−1 dry weight)
Asia Rs306 Rs035 Rubea
Cyanidin3‑diglucoside‑5‑glucoside 0.041 ND 0.235 0.59
Cyanidin3‑(sinapoyl)‑diglucoside‑5‑glucoside 0.105 ND 1.630 0.32
Cyanidin3‑sophoroside‑5‑malonylglucoside 0.032 ND 0.070 ND
Malvidin3‑(p‑coumaroyl)glucoside pyruvic derivative ND ND 0.067 ND
Pelargonidin3‑(6‑acetyl)‑glucoside 0.262 ND 0.256 ND
to perform a syntenic gene search of the A (B. rapa) and C (B. oleracea) genomes from the BRAD database. The Bolbase and EnsemblPlants databases were also searched for anthocyanin genes in these species. A complementary method, Hidden Markov Model profiling, was performed to increase the accuracy of the identified genes within the B. rapa and B. oleracea genomes. The chromosomal position and the sub-genome status of these putative anthocyanin biosynthesis pathway genes, as well as the isoelectric points (Pi), molecular weights, and residual protein size of their predicted proteins, are presented in Additional file 1: Tables S1a and Additional file 2: Table S1b for B. rapa and B. oleracea, respectively.
We identified 73 putative anthocyanin pathway genes
in B. rapa (A genome), of which 72 could be mapped
onto one of its 10 chromosomes; chromosomes A01– A10 contained 3, 12, 12, 7, 10, 4, 6, 1, 12, and 5 putative genes, respectively. The gene, Bra035004, an ortholog of UGT79B1, is located on Scaffold000100 and could not be assigned to a chromosome. Of the other 72 putative genes, 21 were structural biosynthetic genes in the phe-nylpropanoid pathway, 18 were involved in early biosyn-thesis, six were late biosynthesis genes, 12 were positive regulators, 13 were negative regulators, and two encoded transporters (Additional file 1: Table S1a and Additional file 5: Figure S2a).
In B. oleracea (C genome), a total of 58 anthocyanin
biosynthesis pathway genes were identified, of which 37 could be assigned to one of the nine chromosomes; chro-mosomes C01–C09 contained 2, 6, 4, 4, 1, 3, 6, 5, and 6 of the putative genes, respectively (Additional file 1: Table S1b and Additional file 5: Figure S2b). The other 21 genes were present on scaffolds, non-contiguous genomic sequences. The putative B. oleracea genes were categorized as 13 structural biosynthetic genes in the phenylpropanoid pathway, 13 early-biosynthesis genes, six late-biosynthesis genes, 24 positive and negative regu-lators, and two transporters (Additional file 2: Table S1b and Additional file 5: Figure S2b).
Three separate microsynteny maps were constructed using B. rapa, B. oleracea, and A. thaliana orthologous pairs of structural, regulatory, and transporter genes in the anthocyanin pathway to investigate their evolution-ary cues and genetic relationships among the three spe-cies (Fig. 3a–c). Based on our analysis, 41 structural genes
in B. rapa were found to correspond to 24 A. thaliana
orthologous genes, while 32 orthologous gene pairs were recognized between B. rapa and B. oleracea (Fig. 3a). Of the regulatory genes, the 24 identified in B. rapa showed syntenic relationships with 16 orthologous genes in A. thaliana, while 24 pairs of syntenic regulatory genes were found between B. rapa and B. oleracea (Fig. 3b). Mean-while, two syntenic transporter genes corresponding
to an A. thaliana ortholog were identified in both B. rapa and B. oleracea (Fig. 3c). These results suggest that the structural, regulatory, and transporter genes of the anthocyanin pathway maintain close syntenic relation-ships in B. rapa, B. oleracea, and A. thaliana.
Prediction of putative candidate genes for the enhanced accumulation of anthocyanin
Expression profiling of anthocyanin biosynthesis genes We carried out expression profiling of the anthocyanin biosynthesis genes to investigate their role in anthocya-nin accumulation in the leaves of the contrasting Bras-sica lines. No striking effects were observed for the expression of the A-genome (B. rapa) and C-genome (B. oleracea) upstream (PAL1, PAL2, C4H, 4CL1) and down-stream (UGT78D2) genes of the anthocyanin pathway in the four lines examined (Additional file 5: Figure S3a), despite their differences in anthocyanin accumulation. However, the downstream gene UGT78D2 was upregu-lated in the A genome of Rs035, with a seven-fold higher expression than in the green line Rs306. This result is expected because of UGT78D2 gene has a preference of Cyanidin3-(sinapoyl)-diglucoside-5-glucoside metabo-lism and found higher amount in Rs035 compared to Rubea (Table 1).
Among the upstream genes, CHS, CHI, F3′H, and FLS1 showed 2-, 10-, 1.8-, and 20-fold higher levels of expres-sion in the A genome of Rs035 than in its C genome, respectively, and were even 1.5-, 1.8-, 2-, and 10-fold more highly expressed than in the A-genome donor parental line, Asia (Fig. 4a). By contrast, F3H gene showed 1.6-fold higher expression in the C-genome of Rs035 than its A-genome. These results indicate that the highly upregulated upstream genes in the A and C genomes of Rs035 could be a major factor in its high accumulation of anthocyanin, although the genes from both the A and C genomes were upregulated compared to the donor par-ents, indicating a synergistic or positive effect of the two genomes overall. In contrast, the upstream gene F3H in the C genome of Rs035 showed a 1.5-fold higher level of expression than that of in A-genome, and a 15-fold higher expression than in the C-genome donor line, Rubea (Fig. 4a). The upstream gene F3′H in the A genome of Rs035 had twofold higher expression compared to A-genome donor line, Asia. These results indicate that
F3H and F3′H had a strong positive effect on enhancing
the accumulation of anthocyanin accumulation in Rs035, but A and C genomes complemented each other.
and 1.5-fold more highly in Rs035, respectively, than in the A-genome donor parent, Asia (Fig. 4b). Twelve bio-synthetic genes (PAL1, PAL2, 4CL1, CHS, CHI, F3H,
F3′H, UGT78D2, FLS1, DFR, ANS, and UGT75C1)
showed a transgressive overexpression (significantly exceeding the expression levels of both parental species) in the resynthesized line Rs035 in A genome background (Fig. 4a, b and Additional file 5: Figure S3a). Moreover, eight structural and biosynthetic genes (PAL1, PAL2,
C4H, CHS, F3′H, FLS1, DFR, and ANS) showed higher
expression in the resynthesized line Rs035 in C genome background compared to Rs035 in A genome back-ground (Fig. 4a, b and Additional file 5: Figure S3a). The other structural genes of the A and C genomes showed non-additive expression. These results indicate that the highly expressed downstream genes (DFR, ANS, and UGT75C1) might be responsible for the enhanced accu-mulation of anthocyanin in Rs035, and that the expres-sion of these genes is significantly upregulated by the interaction between the A and C genomes.
Expression profiling of regulatory and transporter genes We next attempted to better understand the molecular mechanisms by which R2R3-MYB, bHLH, and WD40 transcription factors regulate anthocyanin biosynthe-sis in the line Rs035. To investigate whether any of the regulatory or transporter genes were upregulated to control the expression of the anthocyanin structural genes or affect anthocyanin accumulation, we performed an expression analysis of two independent regulatory R2R3-MYB genes (MYB12 and MYB111), four genes of the MBW complex [the MYBs PAP2 and MYB113, the bHLH TT8, and the WD40 Transparent Testa Glabra 1 (TTG1)], and one transporter gene (TT19) from both the A and C genomes. Among these seven genes, MYB12,
PAP2, MYB113, and TTG1 from the A and C genome
backgrounds of Rs035 did not show any striking differ-ence in expression, although the A genome genes were quantitatively more highly expressed than those of the C genome, and were even higher than in the A genome donor parental line, Asia (Additional file 5: Figure S3b). In contrast, the regulatory gene MYB111 and the trans-porter gene TT19 in A genome background were signifi-cantly more highly expressed in Rs035 than MYB111 and TT19 in C genome background (170-fold and 75-fold higher expression, respectively). TT8 of C genome showed a 15-fold higher expression level than TT8 of A genome in Rs035 (Fig. 4c). In addition, five regulatory and transporter genes, of which four from A genome (MYB12, PAP2, MYB111 and BrTT19) and one from C genome (TT8) showed transgressive overexpression and two genes (MYB113 and MYB111) of C genome showed transgressively repressed expression in the line Rs035, while one regulatory gene (MYB12) of C genome showed additive expression in Rs035 (Fig. 4c and Additional file 5: Figure S3b). These results also revealed that the interac-tion of the A and C genomes led to synergistic or additive effects on gene expression, possibly due to the comple-mentary actions of the syntenic homeologous regulatory and transporter genes.
Associations between the constituent anthocyanins and the expression levels of the biosynthetic and regulatory genes
A principal component analysis of the total and constitu-ent levels of anthocyanins and the expressions of the bio-synthesis pathway genes in four contrasting lines (Asia, Rs306, Rs035, and Rubea) led to the identification of five principal components (PCs) with an Eigenvalue greater than unity (data not shown). The first three PCs (PC1, PC2, and PC3) explained 97% of the total variance in anthocyanin content (67.5%, 22.5%, and 7% of the vari-ance was explained by PC1, PC2, and PC3, respectively; Table 2). The total variation accounted for by PC1 was
manifested by the higher positive coefficients of some of the upstream and all of the downstream biosynthesis and regulatory genes, the total anthocyanin content, and the majority of the individual anthocyanin components, versus the higher negative coefficients of two upstream biosynthesis genes and one regulatory gene (Table 2 and Fig. 5). PC1 clearly distinguished the red resynthe-sized line (Rs035) from the green line (Rs306) and the two parental lines (Asia and Rubea). This distinction cor-responded to the highly expressed genes, anthocyanin components, and total anthocyanin contents of the four samples, as evidenced by their mean PC scores and dif-ferent positions in the PCA biplot (Table 2 and Fig. 5). The highly expressed biosynthetic and regulatory genes were plotted against the total anthocyanin content and most of the anthocyanin components in the high-antho-cyanin line, Rs035, as shown in the PCA biplot (Fig. 5).
Meanwhile, PC2 clearly separated the resynthesized line Rs035 from its diploid parents B. rapa (Asia) and B. oleracea (Rubea) in the PCA biplot, along with their respective gene expression patterns, total anthocyanin contents, and anthocyanin compositions (Fig. 5). This was further evidenced by the mean PC scores in the resynthesized lines, Rs035 and Rs306 (+ 6.45 and − 3.31, respectively), and the diploid parental lines, Rubea and Asia (− 3.72 and 0.57, respectively; Table 2), and by the higher significant positive correlations between total anthocyanin and the gene expression patterns, as observed from the Pearson’s correlation analysis (Addi-tional file 3: Table S2).
The anthocyanins are a group of flavonoid secondary metabolites with a wide range of colors, from light yel-low to blue or even black. Brassica vegetables have diverse anthocyanin contents; however, very few high-anthocyanin cultivars are available for B. napus. Previ-ously, only one report has investigated the quantitative trait loci regulating anthocyanin accumulation in this species . Here, we attempted to develop resynthe-sized B. napus lines with enhanced levels of anthocyanin in the leaves by hybridizing high-anthocyanin varieties
of B. rapa var. pekinensis (A genome) and B. oleracea
the newly resynthesized allopolyploids [39, 41–47]. Sev-eral studies on resynthesized polyploids (including in the genera Arabidopsis, Brassica, Gossypium, Nicotiana, Triticum, and Triticale) and natural polyploid species
have demonstrated a variety of genomic changes, includ-ing deletions, gene conversions, transposon activa-tions, chromosomal rearrangements, and epigenetic Table 2 Component loadings and mean principal component (PC) scores, showing the association between the total and constituent anthocyanin contents and the expression levels of the biosynthetic and regulatory genes, as determined using a principal component analysis
SD standard deviation, C3-dig5-gl cyanidin 3-diglucoside-5-glucoside, C3-s-dig5-gl cyanidin 3-(sinapoyl)-diglucoside-5-glucoside, C3-sop5-m.gl cyanidin
3-sophoroside-5-malonylglucoside, Mal3-(p-c)gl py de malvidin-3-(p-coumaroyl)glucoside pyruvic derivative, Pe3-(6-ac)-gl pelargonidin-3-(6-acetyl)-glucoside, C3-(p-c) digl5-gl cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside
Letters in mean PC score against the genotypes are mean separation using Tukey test with p < 0.05
Variable PC1 PC2 PC3
PAL1 0.14 0.29 0.24
PAL2 − 0.06 0.36 0.26
C4H − 0.09 0.34 0.24
4CL1 − 0.04 0.37 0.25
CHS 0.22 − 0.01 − 0.19
CHI 0.23 0.01 − 0.09
F3H 0.21 − 0.05 0.19
F3´H 0.23 − 0.08 − 0.03
FLS1 0.23 0.02 0.11
DFR 0.23 − 0.03 0.15
ANS 0.23 − 0.08 0.12
UGT75C1 0.22 − 0.01 − 0.17
UGT78D2 0.23 0.03 0.10
MYB12 0.23 0.04 − 0.05
MYB111 0.22 − 0.03 − 0.22
PAP2 0.22 0.08 0.10
MYB113 0.23 0.01 − 0.05
BrTT8 − 0.11 − 0.34 0.17
TTG1 0.20 0.11 − 0.15
TT19 0.22 − 0.03 0.21
C3‑dig5‑gl − 0.04 − 0.38 0.23
C3‑s‑dig5‑gl 0.21 − 0.10 0.28
C3‑sop5‑m.gl 0.23 − 0.01 − 0.07
Mal3‑(p‑c)gl py de 0.21 − 0.03 0.28
Pe3‑(6‑ac)‑gl 0.20 0.01 − 0.37
C3‑(p‑c)digl5‑gl − 0.12 − 0.33 0.15
Total anthocyanin 0.09 − 0.35 0.23
Eigenvalue 18.22 6.07 2.10
% of the variation explained 67.5 22.5 7.0
Cumulative % of the variation explained 67.5 90.0 97.0
Genotype Mean PC score ± SD
Asia 0.57 ± 0.09 B 0.36 ± 0.12 B − 2.37 ± 0.20 C
Rs306 − 3.31 ± 0.02 C 3.28 ± 0.11 A 0.89 ± 0.09 A
Rs035 6.45 ± 0.04 A − 0.29 ± 0.08 C 0.96 ± 0.08 A
modifications, all of which altered the phenotypes of the resulting plants [48–50].
By using HPLC, we identified five anthocyanin pounds in Rs035, whereas only four and three com-pounds were detected in Asia and Rubea, respectively (Table 1). The additional anthocyanin compound in Rs035 might be the result of recombination and/or a genomic restructuring of the anthocyanin biosynthetic and regulatory genes between its A and C genomes. The genomes of allopolyploids can be considerably different from those of the parental genomes [51–53].
We identified 73 and 58 genes involved in the antho-cyanin pathway in B. rapa (A genome) and B. oleracea (C genome), respectively. The A-genome genes were distributed across all 10 A-genome chromosomes (A01– A10) and the C-genome genes were located on all nine C-genome chromosomes (C01–C09). Some of the genes could not be mapped to chromosomes and were instead mapped to scaffolds; more C-genome than A-genome genes were mapped to these non-contiguous genomic sequences (Additional file 1: Table S1a, Additional file 2: Table S2b and Additional file 5: Figure S2a, b). Simi-lar distributions of anthocyanin genes were previously reported , and whole-genome analyses established that the degree of gene density on the chromosomes might be the result of genome triplication events in
Bras-sica [54, 55]. The genomes of B. rapa and B. oleracea
therefore contain more than one copy of most antho-cyanin biosynthesis genes. The microsynteny analyses
among the anthocyanin biosynthesis and regulatory genes in A. thaliana, B. rapa, and B. oleracea corrobo-rated the close relationships among them, and confirmed their evolution from a common ancestor, consistent with the report on BrAQP genes by Kayum et al. .
Anthocyanins are important secondary metabolites that play a defensive role against a range of abiotic and biotic stresses in plants. Recent studies have also reported multiple benefits of anthocyanin consumption in human health. Despite their importance, the genes involved in anthocyanin biosynthesis and its regulation are poorly understood in B. napus, although they have been well characterized in other Brassicaceae species, including Arabidopsis , Chinese cabbage , cabbage , pak choi (B. rapa ssp. chinensis) , kohlrabi , radish (Raphanus raphanistrum ssp. sativus) , and brown mustard (B. juncea) . Here, we tried to predict the putative candidate gene(s) involved in anthocyanin bio-synthesis, using expression profiling to assess the mag-nitude and nature of their expression patterns. Previous studies reported that MBW transcriptional activation complexes may activate the expression of the anthocya-nin biosynthesis genes [23, 25, 61]. Our expression data showed that the biosynthesis genes PAL, C4H, 4CL, CHS,
CHI, F3H, and F3′H were differentially expressed among
the two parental lines (Asia and Rubea) and the two resynthesized lines (green Rs306 and red Rs035).
Among the upstream genes, A-genome CHS and CHI showed 2- and tenfold higher levels of expression, respec-tively, in Rs035 than the C-genome versions of these genes, while only one C-genome upstream gene, F3H, showed a 1.5-fold higher expression than the A-genome gene (Fig. 4a). The highly expressed upstream genes seem to be putatively involved in the production of flavonols and other flavonoid compounds in the resynthesized line Rs035. Of the downstream genes, FLS1, DFR, ANS, and UGT75C1 from the A- and C-genomes were differen-tially expressed in the examined lines; A-genome FLS1, DFR, BrANS, and UGT75C1 had 20-, 60-, 4-, and 55-fold higher expression levels, respectively, in Rs035 than their C-genome counterparts. Highly expressed genes down-stream of the anthocyanin biosynthesis pathway may contribute to the accumulation of anthocyanin .
Seven upstream (PAL1, PAL2, 4CL1, CHS, CHI, F3H,
F3′H) and four downstream genes (FLS1, DFR, ANS, and
UGT75C1) showed transgressive overexpression in Rs035 in comparison with its parents. Most of the upstream and downstream genes of the Rs035 C-genome showed additive expression (Fig. 4a, b and Additional file 5: Fig-ure S3a). The transgressive overexpression of the above-mentioned genes might be caused by several mechanisms encountered in the hybrid , including the reunion of divergent genomes in the same nucleus, enabling the Fig. 5 Biplot of total and constituent anthocyanin contents and
the expression of anthocyanin biosynthesis pathway genes in four
Brassica lines (Asia, Rubea, Rs035, and Rs306), determined using a principle component analysis (PCA). Genotype text color reflects the visual phenotype of the leaves. Blue and italic texts denote anthocyanin components and gene names, respectively. C3-dig5-gl
cyanidin 3‑diglucoside‑5‑glucoside, C3-s-dig5-gl cyanidin 3‑(sinapoyl) ‑diglucoside‑5‑glucoside, C3-sop5-m.gl cyanidin 3‑sophoroside‑5‑ma lonylglucoside, Mal3-(p-c)gl py de malvidin‑3‑(p‑coumaroyl)glucoside pyruvic derivative, Pe3-(6-ac)-gl pelargonidin‑3‑(6‑acetyl)‑glucoside,
interaction of divergent genes  or a dosage effect of the homeologous genes within the same background.
Based on their significantly higher expression levels in one or both genomes of Rs035 in comparison with the parental lines, we predicted that one R2R3-MYB (MYB111) and one bHLH (TT8) of the MBW complex, as well as a transporter gene (TT19), may be responsible for the higher anthocyanin accumulation in Rs035 (Fig. 4c and Additional file 5: Figure S3b). High anthocyanin accumulation in Rs035 might achieved due to combined and complementary effect of those three regulatory and transporter genes by activating the transcription of struc-tural genes, while one regulatory R2R3-MYB (MYB111) as well one transporter gene (TT19) were upregulated in the parent line Asia; one bHLH (TT8) of the MBW complex was upregulated in the parent Rubea. The com-bined effect of those regulatory and transporter genes might have higher effect on activating the transcription of structural genes in Rs035 than in their parents.
The R2R3-MYB PAP1, MYB113, and MYB114, and the bHLH TT8 are known to be involved in the upregulation of anthocyanin biosynthesis in Arabidopsis leaves . Xie et al.  reported that the bHLH-encoding regu-latory genes and a large proportion of structural genes are candidates for the accumulation of anthocyanins in B. rapa. In A. thaliana and a number of other species, MBW complexes were identified as the activators of anthocyanin biosynthesis [66, 67]. In the resynthesized line Rs035, R2R3-MYB and bHLH transcription fac-tors could bind to specific cis-elements in the promot-ers of the anthocyanin structural genes; this finding is in agreement with . The transgressive expression of the predicted regulatory and transporter genes in the resyn-thesized line Rs035 might also be due to interaction of cis- and trans-regulatory factors in the synthetic hybrids as reported by other research efforts [68–70].
Based on our comparative univariate expression data, we identified three early-biosynthesis (CHS, CHI, and
F3H), four late-biosynthesis (FLS1, DFR, ANS, and
UGT75C1), two transcriptional regulator (MYB111 and TT8), and one transporter (TT19) genes as the putative candidates responsible for the enhanced accumulation of anthocyanins in the resynthesized line Rs035 (Fig. 4a–c and Additional file 5: Figure S3a, b). We deployed a mul-tivariate analytical approach to identify and visualize the overall association of the expression of these genes with the total and constituent anthocyanin contents in the four Brassica lines. The association of the highly expressed genes with the total anthocyanin contents and the anthocyanin components of Rs035 in the PCA biplot indicate that these genes may be responsible for the higher accumulation of anthocyanins in this line. The total anthocyanin contents and the expression of
key regulators (MYB111, TT8, and TT19) showed sig-nificant positive correlations with the expression levels of the structural genes, which further demonstrated the association between these genes and the total anthocya-nin contents. Previous studies indicated that each B. rapa and B. oleracea chromosome has a homologous counter-part in B. napus [71, 72]; therefore, the predicted A- and C-genome genes are likely confined within the resynthe-sized B. napus. A previous study reported the upregu-lation of BrCHS, BrF3H, BrANS, BrDFR, and BrTT8 in anthocyanin-enriched pak choi , while BoF3′H, BoMYB2, and BoTT8 were hypothesized to be respon-sible for anthocyanin accumulation in red cabbage . Our PCA biplot data support the involvement of these genes in the accumulation of high levels of anthocyanin in Rs035 (Fig. 5).
Anthocyanin-enriched B. napus lines were developed by hybridizing anthocyanin-rich varieties of B. rapa and B. oleracea. A series of in silico analyses were used to identify and characterize the anthocyanin genes and determine their chromosomal distributions and syntenic relationships. Variable numbers of anthocya-nin genes are distributed across all chromosomes of the reconstituted B. napus lines, which were inherited from the A- and C-genome donor parents. Antho-cyanin biosynthesis is a complex enzymatic process; therefore, a coordinated analysis of the expression of many anthocyanin biosynthesis-related genes was carried out to elucidate the molecular mechanisms involved, within the limits of the environmental cues that control the pathway and the final accumulation of the end products. An overview of the most highly expressed genes of each of the pathway nodes in our study revealed that the late-biosynthesis genes of the A-genome are comparatively highly expressed in Rs035 line compared with those of the C genome in the same background, but A and C genomes complemented each other. Our expression data also clearly demonstrated that the independent regulator BrMYB111, BolTT8, and the transporter gene BrTT19 are key regulators controlling the production of high levels of antho-cyanin in resynthesized B. napus, as demonstrated by their transgressive and additive expression patterns. Our study lays the foundation for enabling the devel-opment of high-anthocyanin-accumulating cultivars
of B. napus, with brilliant colors and
other commercial rapeseed lines using molecular and conventional breeding.
Plant materials and sampling
Four lines from the genus Brassica were used, includ-ing a red-green Chinese cabbage line (B. rapa cv. Asia), a red cabbage line (B. oleracea cv. Rubea), a green resynthesized rapeseed line (allopolyploid B. napus cv. Rs306), and a red resynthesized rapeseed line (allopoly-ploid B. napus cv. Rs035). The Rs035 line was developed by crossing the inbred Chinese cabbage line B. rapa cv. Asia with the cabbage line B. oleracea cv. Rubea. The embryos were rescued 18–20 days after crossing and were cultured on Murashige and Skoog (MS) medium. The chromosomes of the developed plants were dip-loidized using a 4-hr 0.1% colchicine root treatment. Successfully crossed hybrid plants were identified using the conserved ortholog set (COS) marker COS1078 . The genotypes of the allopolyploid plants with diploid chromosomes were confirmed using a CyFlow ploidy analyzer and 4′,6-diamidino-2-phenylindole (DAPI) solution (Sysmex, Norderstedt, Germany).
RNA extraction and cDNA synthesis
The fourth leaves of 6-week-old plants were collected and immediately frozen in liquid nitrogen, then stored at − 80 °C. Around 2 g of each leaf sample was ground into powder for RNA extraction and anthocyanin quantifica-tion. Total RNA was isolated from 100 mg of the finely ground powder using a RNeasy Plant Mini Kit (Qia-gen, Hilden, Germany), according to the manufacturer’s instructions. The RNA was treated with RNase-free DNase I (Qiagen) before cDNA synthesis. The concen-tration of the extracted RNA was measured using a Nan-oDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was synthe-sized using a First-Strand cDNA Synthesis kit (Thermo Fisher Scientific) in a 20-μl reaction, according to the manufacturer’s instructions, then stored at − 20 °C until required.
Quantification of total anthocyanin content
The total anthocyanin content was determined following a previously described protocol  with some modi-fications. Three leaves of each genotype were frozen in liquid nitrogen and ground into powder, then 100 mg of each sample was transferred into an Eppendorf tube containing 1 ml acidic methanol (1% HCl, w/v). Sam-ples were mixed overnight at room temperature by shak-ing at 50 rpm in the dark. After that, the mixtures were centrifuged at 12,000×g for 10 min. The supernatants
were collected and the absorbance of each sample was determined at 530 and 657 nm wavelengths. The total anthocyanin content was computed using the follow-ing equation: QAnthocyanins= (A530 – 0.25 × A657) × FW−1,
where QAnthocyanins=total anthocyanin content,
A530=absorption at 530 nm, A657= absorption at
657 nm, and FW = fresh weight of leaf samples (g). The total anthocyanins were quantified from five replicates of each biological sample.
Extraction and quantification of anthocyanin compounds using high‑performance liquid chromatography
For the high-performance liquid chromatography (HPLC) analysis, the leaf samples were freeze-dried and ground into a fine powder in liquid nitrogen. A 100-mg sample of each finely powdered leaf was vortexed for 5 min in 2 ml 5% formic acid (v/v) in ultrapure water, after which it was sonicated for 20 min. The samples were centrifuged at 12,000 rpm for 10 min, then the super-natant was filtered through a 0.45-μm PTFE filter (Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The extracted antho-cyanins were quantified using an Agilent 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA), and the chromatographic separation was carried out using reverse phase columns (particle size 4 μm; C18 80A, 250 × 4.60 mm; Phenomenex, Torrence, CA, USA) at a temperature of 40 °C. The injection volume was 10 μl with a flow rate of 1 ml min−1. Elution was performed
using mobile phase A (5% formic acid) and B (acetoni-trile), with the following gradient program: 0–8 min using 5–10% B; 8–15 min at 13% B; 15–18 min using 15% B and kept constant until 25 min. After 25 min, the concentra-tion of B was quickly decreased to 5% and kept constant until 35 min. Detection was carried out at a wavelength of 520 nm. Individual anthocyanin components were quantified by comparing the area under their HPLC peak with that of a known standard (cyanidin-3-0-glucoside), and expressed as mg g−1 dry weight.
Selection and in silico analysis of anthocyanin biosynthesis genes
the primary gene features (gene structure, distribution on chromosomes, gene position, strand, protein length, molecular weight, and iso-electric point) were analyzed using ExPasy (http://au.expas y.org/tools /pi_tool.html). The physical positions of the genes were drafted onto the A- and C-genome chromosomes from B. rapa and B. oleracea, respectively, using Map Chart v.2.2 (http:// www.wagen ingen ur.nl/en/show/Mapch art.htm). The microsyntenic relationships of the anthocyanin struc-tural, regulatory, and transporter genes in A. thaliana, B. rapa, and B. oleracea were plotted using circos software (http://circo s.ca/) .
Expression analysis using quantitative real‑time (qRT) PCR
Gene expression levels were analyzed using qRT-PCR performed on a Roche LightCycler® 96 System (Roche Applied Science, Penzberg, Germany). Gene-specific primers (Additional file 4: Table S3a, b) were designed using an online tool, Primer3Plus , and were tar-geted to the gene sequences collected from BRAD  and Bolbase . The 10-μl reaction mixture contained 5 μl of 2 × qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystems, London, UK), 1 μl of each gene-specific forward (F) and reverse (Rv) primer (10 pmol), 2 μl dis-tilled deionized water (ddH2O), and 1 μl of 80 ng μl−1
cDNA as a template. The qRT-PCR was performed under the following conditions: initial denaturation at 95 °C for 5 min; followed by 50 cycles of 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 15 s. Three replicates of each sample were analyzed. The data were processed using the LightCycler 96 SW 1.1 software and quanti-fied using the Cq value of each qRT-PCR amplicon, following the 2−ΔΔCt method . The data were
nor-malized against the mean amplicon value of the three ACTIN genes (as internal controls) expressed by B. rapa and B. oleracea.
The total and constituent anthocyanin contents and the gene expression levels were analyzed using one-way analysis of variance (ANOVA) and Tukey’s pair-wise comparisons in Minitab v.17 (Minitab Inc., State Col-lege, PA, USA). The data were presented as the mean of three replicates ± standard deviation (± SD). Data used for principal component analyses (PCAs) were stand-ardized by subtracting the mean and dividing the result by the standard deviation. The constituent anthocyanins determined using HPLC and the relative gene expression levels calculated from the qRT-PCR were set as variables for the PCA using Minitab v.17 (Minitab Inc.).
Additional file 1: Table S1a. In silico analysis of Anthocyanin genes iden‑ tified in B. rapa with their Arabidopsis orthologs and biological activity.
Additional file 2: Table S1b. In silico analysis of anthocyanin genes iden‑ tified in B. oleracea with their Arabidopsis orthologs and biological activity.
Additional file 3: Table S2. Pearson’s correlation analysis among the expression of the anthocyanin biosynthesis pathway genes of A‑ and C‑genome along with anthocyanin components in the four contrasting Brassica lines.
Additional file 4: Table S3a. Oligonucleotide primers used for qRT–PCR analysis of B. rapa.Table S3b. Oligonucleotide primers used for qRT–PCR analysis of B. oleracea.
Additional file 5: Figure S1. Morphological distinctiveness and anthocy‑ anin contents of four Brassica lines. The visual phenotype (a) and 4th‑leaf total anthocyanin contents of 6‑week‑old plants (b) of the parental lines Asia (B. rapa) and Rubea (B. oleracea), the red allopolyploid resynthesized B. napus (Rs035), and the control green allopolyploid resynthesized B. napus (Rs306) are shown. Figure S2. Chromosomal distributions of the putative anthocyanin biosynthesis genes in (a) Brassica rapa and (b) Brassica oleracea. The chromosome number is indicated at the top of each chromosome. The scale (left) is in megabases (Mb). The colors represent the putative structural genes of the phenylpropanoid pathway (black), early‑biosynthesis (red), late‑biosynthesis (green), positive‑regulatory (blue), and negative‑regulatory (brown) genes. Genes assigned to scaffold sequences are not shown on the physical maps. Figure S3. Relative gene expression of anthocyanin pathway genes which did not show striking expression changed compared among the studied four lines, (a) structural and (b) regulatory and transporter genes.
4CL: 4‑coumaratel‑coA ligase; ANOVA: analysis of variance; ANS: anthocyanidin synthase; bHLH: basic helix loop helix; Bolbase: Brassica oleracea Genomics Database; BRAD: Brassica Database; C4H: cinnamate‑4‑hydroxylase; cDNA: complementary DNA; CHI: chalcone isomerase; CHS: chalcone synthase; COS: conserved ortholog set; DFR: dihydroflavanol reductase; F3H: flavanone‑3‑hy‑ droxylase; FLS: flavonol synthase; FW: fresh weight; HPLC: high performance liquid chromatography; MS: Murashige and Skoog; MYB: myeloblastosis family of transcription factors; PAL: phenylalanine ammonia lyase; PCA: principal component analysis; Pi: isoelectric point; qPCR: quantitative polymerase chain reaction; Rs: resynthesised; RT‑PCR: reverse transcription polymerase chain reaction; SD: standard deviation; TAIR: the arabidopsis information resource; TT: transparent testa; TTG1: transparent testa glabra 1; UGT : uradin glycosyltransferase.
I‑SN, J‑IP, and UKN formulated the concept and designed the research. GG performed the research, analyzed the real‑time expression data, and prepared the tables and figures. UKN conducted the statistical analysis and wrote the manuscript. MRH conducted the PCA and polished the manuscript. MKB and H‑TK performed the in silico analysis. HRK conducted the HPLC analysis. All authors read and approved the final manuscript.
1 Department of Horticulture, Sunchon National University, 255 Jungang‑ro,
Suncheon, Jeonnam 57922, South Korea. 2 Department of Genetics and Plant
Breeding, Bangladesh Agricultural University, Mymensingh, Bangladesh.
3 University‑Industry Cooperation Foundation, Sunchon National University,
255 Jungang‑ro, Suncheon, Jeonnam 57922, South Korea. 4 Plant Systems
Engineering Research Center, Korea Research Institute of Bioscience and Bio‑ technology, Daejeon, South Korea.
The authors declare that they have no competing interests.
Availability of data and materials
We declare that the dataset(s) supporting the conclusions of this article are included within the article and its Additional files 1, 2, 3, 4, 5.
Consent for publication Not applicable.
Ethics approval and consent to participate Not applicable.
This research was supported by the Golden Seed Project (Center for Horticul‑ tural Seed Development, No. 213007‑05‑3‑CG100); the Ministry of Agriculture, Food and Rural Affairs (MAFRA); the Ministry of Oceans and Fisheries (MOF); the Rural Development Administration (RDA); and the Korea Forest Service (KFS), South Korea.
Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations.
Received: 18 July 2018 Accepted: 9 November 2018
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